Ribosome display

ABSTRACT

The use in a ribosome display system for selection of a specific binding pair member (e.g. antibody molecule) able to bind a complementary specific binding pair member (e.g. antigen) of encapsidating specific binding member/ribosome complexes in a viral coat, optionally in combination with incorporation of an Midvariant RNA template and optionally one or more other improvements selected from: a glycine-serine tether, protein disulphide isomerase, protein disulphide isomerase in combination with oxidised and reduced glutathione at a ratio of between 1:1 and 10:1, addition of oxidised and reduced glutathione at a ratio of between 1:1 and 10:1 after 30 minutes of in vitro translation; blocking with heparin during selection.

This is a divisional of U.S. application Ser. No. 09/817,661, filed Mar.26, 2001, which claims the benefit of U.S. Provisional PatentApplication No. 60/193,802 filed on Mar. 31, 2000.

The present invention relates to ribosome display and is based invarious aspects on various improvements made by the present inventorswhich individually or in combination provide advantages over existingtechniques.

Ribosome/polysome selection involves construction of nucleic acidlibraries, screening for binding, and identification of binding entitiesof interest. The library is made by synthesising a DNA pool of diversesequences that are then transcribed to produce a pool of mRNAs. In vitrotranslation is used to generate the encoded polypeptides or proteinsdisplayed on the ribosomes, and desirable binding interactions areselected using immobilised target antigen. mRNA encoding the bindingentities can be recovered and used to make cDNA, which can then beamplified and the process may be repeated to enrich the population forgenes encoding binders. The selected proteins may later be identified bycloning individual coding sequences and DNA sequencing.

Recovery of mRNA from polysome complexes was first reported in 1973 in apaper describing a protocol to capture mRNA coding for a mouseimmunoglobulin L-chain using antibodies and immobilised oligothymidine(Schechter (1973) PNAS USA 70, 2256-2260). Improvements to the polysomeimmunoprecipitation protocols were made by Payvar and Schimke (Eur. J.Biochem. (1979) 101, 271-282) and cDNA clones for the heavy chain ofHLA-DR antigens were obtained after immunoprecipitation of polysomesusing a monoclonal antibody (PNAS USA (1982) 79, 1844-1848). Productionof libraries of antibodies by ribosome display was proposed and patentedby Kawasaki (U.S. Pat. No. 5,643,768 and U.S. Pat. No. 5,658,754).

There have been various examples of the use of ribosome display usingeither eukaryotic or prokaryotic translation systems. The firstdemonstration of selection of peptide ligands that using an E. coliextract was by Mattheakis et al., (PNAS USA (1994) 91, 9022-9026 andMethods Enzymol (1996) 267, 195-207). This group demonstrated selectionof peptide ligands that are similar to known peptides epitopes of agiven antibody, using the antibody as a selection substrate.High-affinity peptide ligands which bind prostate-specific antigen havebeen identified using polysome selection from peptide libraries using awheat germ extract translation system (Gersuk et al., (1997) Biotech andBiophys. Res. Com. 232, 578-582). The selection of functional antibodyfragments was reported using an E. coli translation system designed forincreased yield of ternary complexes and allowing disulphide bondformation (Hanes and Pluckthun, PNAS USA (1997) 94, 4937-4942). Thisexperimental set up has subsequently been used to select antibodies froma murine library, and it was shown that affinity maturation occursduring the selection due to the combined effect of PCR errors andselection. A scFv fragment with a dissociation constant of about 10-¹¹Mwas obtained (Hanes et al., PNAS USA (1998) 95, 14130-50). Enrichmentfor specific of antibodies from mixed populations using rabbitreticulyocyte lysate extracts has also been demonstrated (He and Taussig(1997) NAR, 5132-5234).

The technology as reported in the literature has not been applied to theselection of antibodies that bind to a target antigen directly from anaïve library. To date the libraries created have been generated usingmaterial from mice immunised with a particular antigen and suchlibraries have formed the basis of affinity selection procedures. Anumber of factors may contribute to the difficulty in generatingantibodies directly from naive libraries. These include the following.

(1) The generation of polysomes using a ribosome display library willresult in a number of ribosomes translating the same mRNA, only one ofwhich will be able to completely translate the entire message. Theremaining ribosomes will partially translate the message and stall sinceribosome release from the end of the message does not occur in thissystem. This may result in the expression of partial polypeptidefragments that may not be capable of forming correct secondary andtertiary structures. These partially translated fragments may haveexposed hydrophobic surfaces and as a result may be non-specificallysticky. The presence of partially translated polypeptide fragments in aselection system may result in the non-specific selection of partiallytranslated fragments, and may reduce the efficiency of the selectionprocess. The present inventors have generated populations of monosomesrather than polysomes, which by definition avoid the presence ofpartially translated protein. A single ribosome is recruited to a singlemRNA molecule, so producing one full length displayed polypeptide permRNA.

(2) Polypeptides may not be correctly folded in the in vitro translationsystem and hence not be able to correctly interact with their bindingpartner (e.g. antibody molecules may not be able to fold and interactcorrectly with antigen). Proteins fold through intermediate states thathave exposed hydrophobic surfaces that have a tendency to aggregate. Inaddition, proteins can misfold by incorrect disulphide formation. It isthought that intramolecular disulphide bond formation may be the ratelimiting step in the folding of some proteins. The process may involvethiol S—S interchange reactions in which-incorrectly linked S—S bridgesare replaced by native S—S bonds. It has also been shown that correctlyS—S bonding in newly synthesised protein in rabbit reticulocyte lysatesdepends on the relative amounts of oxidised and reduced thiols(Kaderbhai and Austen 1985, Eur J. Biochem, 153, 167-178). The extent ofcorrect S—S pairing is dependent on the amounts of GSSG added at theonset of translation and is also dependent on the rates at which thiollevels change during the translation phase. The inventors have providedan approach which can be used to achieve increased levels of correctlyfolded and therefore active antibody molecules (e.g. scFv) in eukaryoticribosome display systems, employing protein disulphide isomerase (PDI).

(3) Naive libraries used as starting points for antigen selectionsshould be highly diverse and incorporate various features includingdetection tags and tethers to avoid steric hindrance between theribosome and the scFv and to allow efficient co-translational folding ofthe scFv. To date, antibody libraries generated for use in ribosomedisplay selections have been produced from immunised mice and clonedinto specifically designed ribosome display vectors (Hanes et al., PNASUSA 1998, 95: 14130-50). It has, however, also been shown thatconstruction and in vitro expression of an antibody library presented asa population of PCR fragments, rather than cloned into a ribosomedisplay expression vector, is possible (Makeyev et al., FEBS Letters 444(1999) 177-180). Makeyev et al. describe generation of a scFv repertoireusing PCR assembly of VH and VL gene segments with a linker fragment.The use of PCR assembly techniques to generate naïve repertoires avoidsthe need for cloning and hence theoretically allows generation of verylarge libraries.

The present inventors have designed a novel cloning-independent PCRassembly strategy to generate ribosome display libraries containingfeatures necessary for transcription and translation at the 3′ end ofthe fragment, and using a DNA cassette approach to incorporate tetherfragments at the 5′ end of the construct. Very large libraries can begenerated with a choice of tether cassette suitable to the particularlibrary or application. Tethers fragments can be structured,non-structured, contain packaging or replication sequences, or be usedas the basis for generation of affinity maturation libraries byextension of the tether into the polypeptide to be displayed. In thiscase a mutagenic oligonucleotide is designed to amplify the tetherfragment along with the region of the polypeptide (e.g. VH CDR3 for ascFV molecule) which is to be targeted for mutation. The mutagenesisoligo should cover the region of mutation and also incorporate an anchorregion upstream of the region of mutagenesis to allow efficient OCRassembly of the complete polypeptide tether construct. Examples ofstrategies for use of tether cassette are outlined in FIG. 1. Thepresent inventors have designed a novel mutagenesis strategy thatenables the mutagenesis steps to be incorporated into a ribosome displayselection cycle. Selected mRNA may be subjected to mutagenesis, prior toRT-PCR, using a primer designed to target sequence encoding a particularregion of the polypeptide or peptide (e.g. CDR of an antibody molecule).Following assembly and pull-through reactions, PCR products may then beused directly in the next round of selection; enabling isolation ofbinding molecules with improved binding capabilities at each stage. Thismay be used to mutagenise all the CDR's of an antibody molecule. (FIGS.7, 8 and 9 illustrate embodiments of this aspect of the presentinvention.)

(4) The RNA displayed in the ribosome display system is labile and proneto degradation. An RNase-free environment must be provided at all timesduring the selection procedure and preferably work must be carried outat 4° C. It is also a requirement that the antigen on which selectionsare being carried out is free of any RNase contamination and is highlypurified. These constraints limit the applicability of ribosome displayselection.

The present inventors have developed a method for encapsidating ribosomedisplay RNA in a protein coat which greatly increases the stability ofRNA over a range of temperatures, and renders it resistant todegradation to RNase.

In preferred embodiments, the protein used for encapsidation is tobaccomosaic virus (TMV) coat protein, but other plant or animal viral coatproteins may be employed.

Other plant or animal viral coat proteins may be employed. Variousrod-shaped or bacilliform plant viruses have the ability to selfassemble and these systems may be applied to provide packaging reagentsfor ribosome display libraries (Hull and Davies (1983) Geneticengineering with plant viruses and their potential as vectors In“Advances in Virus Research” (Lauffer and Maramorosch, eds) Vol. 28,1-33. Academic Press, Orlando, Fla.). Such viruses include any positivestrand plant or animal RNA virus. Plant RNA viruses include, but are notlimited to, members of the Tobamovirus, Potexvirus, Potyvirus,Tobravirus, Cucumovirus or Comovirus families. Animal viruses wouldinclude, but are not limited, to the Togaviridae, Flaviviridae,Picornaviridae and Caliviridae. Coat proteins derived from DNA virusessuch as cauliflower mosaic virus may also be employed for encapsidatingDNA or RNA, as may coat proteins or bacteriophage lambda (reviewed in“Principles of Gene Manipulation” Third edition (1985) Old and Primrose,Blackwell, Oxford).

Native TMV particles are extremely stable and retain infectivity fordecades. Over 2100 copies of a 17.6 kDa coat protein fully protect the6.4 kb single stranded RNA genome against degradation. TMV was employedin spontaneous self-assembly of multimeric biological structure in vitro(Butler and Klug 1971 Nature New Biol. 229, 47-50). TMV assembly isinitiated by a specific interaction between a prefabricated (20 S)protein aggregate (the disk, or protohelix) and a stem-loop-structuredRNA origin of assembly sequence (OAS) sequence, that is centred either0.4 kb or 0.9 kb from the 3′ end of the genomic RNA (Zimmern and Wilson,1976 FEBS Lett 71, 294-298). In addition to native TMV RNA, TMV coatprotein has also been shown to package chimeric ssRNAs efficiently(Sleat et al., 1986, Virology 155, 299-308) in a length- andsequence-independent manner provided that a contiguous region of theloop 1 of the OAS sequence (genome co-ordinates 5444-5518) was present.This can occur in vitro, or in vivo in transgenic tobacco plants (Sleatet al., 1988, NAR 16, 3127-3140). Only ssRNA, not ssDNA can be packed bythe coat protein. It has been suggested that the exceptional stabilityof TMV-like particles to proteases and RNases can be exploited torecover, store, and protect otherwise labile mRNA molecules to deliverthem into plant or animal cells for subsequent cotranslationaldisassembly (Gallie et al., Science 236, 1122-1124).

The present inventors have now shown that it is possible to generateribosome display libraries containing the a viral packaging OAS whichcan be encapsidated in viral coat protein, hence providing increasedstability to the RNA. TMV is co-translationally disassembled in vivo(Wilson 1984, Virology, 137, 255-265) and in vitro. Encapsidatedribosome display RNA may also be co-translationally disassembled. It hasalso been demonstrated that the presence of translocating ribosomespresent on the OAS-containing RNA do not completely inhibit RNApackaging. It appears that the RNA molecule is packaged in the 3′ to 5′direction as far as the progressing ribosome, but eventually thepackaging process is blocked by the advancing ribosome. Completetranslation of the ribosome display mRNA may be allowed to generate thepolypeptide mRNA ribosome complex prior to initiation of the packagingreaction to produce a fully encapsidated complex.

The inventors have additionally incorporated into ribosome displayconstructs and methods an RNA template known as Midvariant (MDV) RNA,enabling replication by Qβ replicase (Wu et al., PNAS, 1992, 89:11769-73. This allows for exponential replication of a ribosome displaylibrary in vivo. For replication by Qβ replicase, the RNA templateadopts a secondary structure to initiate recognition and replication.Brown et al, Biochemistry, 1995, 34:45, 14765-74 have shown two RNAbinding sites on Qβ replicase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a scheme for generation of a PCR assembled naive scFvrepertoire in accordance with an embodiment of the present invention.Primers PEU, Mycseq 10 and Hismyc Back are as described in the text. Inpreferred embodiments of the invention, the tether ispoly-glycine-serine. In the assembled construct, PEU is the proteinexpression unit consisting of promoter, consensus sequence and ribosomebinding site.

FIG. 2 illustrates a ribosome display construct according to anembodiment of the present invention:

FIG. 2A illustrates key features: the protein expression unit (PEU)consists of T7 promoter, Kozak consensus sequence, ribosome bindingsite. Various cloning sites are included, as illustrated, and a sequenceencoding a tether.

FIG. 2B shows the sequence of the ribosome display construct of thisembodiment of the present invention (within a pCU vector). The boldtriplets show the SfiI, PstI and NotI restriction sites. The continuousstretch of bold shows the sequence encoding the tether. The key featuresof the construct of this embodiment of the present invention are a T7promoter, ribosome binding site, Kozak consensus sequence,SfiI/PstI/NotI cloning sites, his tag, myc tag, gly-ser tether and HAtag.

FIG. 3 shows the effect of redox state on activity of an antibodymolecule (anti-TNFα scFv). A range of different ratios ofoxidised:reduced glutathione was added to coupled reactions of ananti-TNFα scFv and tested on TNFα or BSA as a control. 1=buffer only;2=10:1 oxidised:reduced; 3=1:1 oxidised:reduced; 4=1:10oxidised:reduced; 5=reduced only.

FIG. 4 ilustrates the effect of PDI concentraion on anti-TNFα scFvactivity. Coupled reactions were performed with the antibody on TNFα orBSA as a control, at various titration concentrations of PDI (μg/ml).

FIG. 5 illustrates a ribosome display construct with the addition of TMVOAS (origin of assembly sequence) for packaging, in accordance with anembodiment of the present invention.

FIG. 6 illustrates a ribosome display construct with the addition of TMVOAS (origin of assembly sequence) for packaging, and MDV sequences assubstrate for replicase, in accordance with an embodiment of the presentinvention.

FIG. 7 illustrates a scheme in accordance with an embodiment of thepresent invention for targeted mutagenesis of a CDR of an antibodymolecule VL domain, integrated into a ribosome display selection cycle.

FIG. 8 illustrates a scheme in accordance with an embodiment of thepresent invention continuing on from the scheme illustrated in FIG. 7and showing targeted mutagenesis of additional CDR's, integrated into aribosome display selection cycle.

FIG. 9 illustrates a scheme in accordance with an embodiment of thepresent invention for successive mutagenesis of CDR's of an antibodymolecule in a ribosome display selection cycle.

According to various aspects, the present invention provides the use ofany one or more of the following features in a ribosome display systemfor selection of a specific binding pair member (e.g. antibody molecule)able to bind a complementary specific binding pair member (e.g.antigen):

(i) a glycine-serine tether, in a eukaryotic or prokaryotic system;

(ii) protein disulphide isomerase (PDI), in a eukaryotic system;

(iii) protein disulphide isomerase (PDI) in combination with oxidisedand reduced glutathione at a ratio of between 1:1 and 10:1, in aeukaryotic or prokaryotic system;

(iv) addition of oxidised and reduced glutathione at a ratio of between1:1 and 10:1 after 30 minutes of in vitro translation in a eukaryotic orprokaryotic system;

(v) blocking with heparin during selection, in a prokaryotic oreukaryotic system, especially when using yeast t-RNA;

(vi) encapsidating sbp member/ribosome complexes in a viral coat, e.g.TMV coat, in a prokaryotic or eukaryotic system;

(vii) encapsidating sbp member/ribosome complexes in a viral coat, e.g.TMV coat, in combination with incorporation of an MDV RNA template, in aprokaryotic or eukaryotic system

(viii) employing a mutagenic primer in RT-PCR generation of a DNA copyof the ribosome display library prior to a further round of selection,in a eukaryotic or prokaryotic system.

Any one or more of features (i) to (viii) may be employed in differentaspects and embodiments of the present invention.

A method according to the present invention may be a method of obtaininga member of a specific binding pair (sbp) that binds a complementary sbpmember of interest, the method comprising:

(a) providing mRNA molecules, each mRNA molecule comprising a nucleotidesequence encoding a specific binding pair member and lacking an in-framestop codon;

(b) incubating the mRNA molecules under conditions for ribosometranslation of the mRNA to produce the encoded specific binding pairmember, whereby complexes each comprising ribosome, mRNA and encodedspecific binding pair member displayed on the ribosome are formed;

(c) bringing the complexes into contact with the complementary sbpmember of interest, and selecting one or more complexes displayingspecific binding pair member able to bind the complementary sbp memberof interest under the conditions of the selection.

RNA from a selected complex or complexes may be isolated and/or used inprovision of DNA, which DNA may be used in production of the encodedspecific binding pair member and/or employed in a further round ofselection using ribosome display (or less preferably in this contextbacteriophage display).

Generally a library, population or repertoire of diverse mRNA sequencesis provided, encoding a library, population or repertoire of diversepeptides or polypeptides with the potential to form specific bindingmembers.

The ribosome translation system employed may be prokaryotic oreukaryotic. Both have been successfully used in the art for display andselection of a number of different binding molecules. See for example:Mattheakis et al., (1994) PNAS USA 91, 9022-9026; Mattheakis et al.,(1996) Methods Enzymol 267, 195-207; Gersuk et al., (1997) Biotech andBiophys Res Com 232, 578-582; Hanes and Pluckthun (1997) PNAS USA 94,4937-4942; Hanes et al., (1998) PNAS USA 95, 14130-50; He and Taussig(1997) NAR 5132-5234.

A construct for ribosome display may comprise a RNA polymerase promoter(e.g. T7 polymerase promoter), ribosome binding site, Kozak consensussequence, initiation codon and coding sequence of polypeptide, peptideor protein. One or more nucleotide sequences encoding one or moredetection tags may be included to provide for production of apolypeptide, peptide or protein further comprising one or more detectiontags (e.g. histidine tag). One or more features providing a featureaccording to the present invention or any combination thereof may beincorporated into a construct according to the present invention asdisclosed herein.

A DNA construct may be cloned into any suitable plasmid or vector, e.g.pUC.

In accordance with one aspect of the invention, a method as outlinedabove is provided in which any one or more of features (i) to (viii)above is included (alone or in combination of any two or more, e.g. 3,4, 5, 6, 7 or 8).

Thus, in one aspect the present invention provides a method comprisingsteps (a), (b) and (c) as indicated, wherein each mRNA moleculecomprising a nucleotide sequence encoding a specific binding pair memberand lacking an in-frame stop codon further comprises sequence encoding agly-ser tether to provide a fusion of encoded specific binding pairmember and tether displayed on the ribosome surface. The tether may beprovided C-terminally to the encoded specific binding pair member. Apreferred poly-gly-ser tether for use in accordance with the presentinvention may comprise or consist of about 24 glycine-serine (GS) units,10-50 GS units, 10-20, 10-30, 20-30, 20-40, 22, 23, 24, 25, 25 or 27 GSrepeats. Other preferred tethers comprise 1-12 glycine-serine units. Thetether may include one or more additional amino acids or tags at eitherend or both ends.

Experimental examples included below demonstrate that a gly-ser tethercan be used in ribosome display. The number of specific antibodiesgenerated by incorporation of a GS tether may be greater than inexperiments that are identical except in use of a gene III based tetherinstead.

In a related aspect, the present invention provides a nucleic acidconstruct (DNA or RNA) for ribosome display comprising a nucleotidesequence encoding a glycine-serine (usually poly-glycine-serine) tether.Such a construct generally comprises additional features for ribosomedisplay.

In embodiments of the present invention employing other aspects where aglycine-serine tether is not employed, a standard tether may beemployed, e.g. using a domain of gene III of a filamentous bacteriophage(Hanes and Pluckthun, PNAS USA 1997, 94:4937-4942) or a kappa lightchain constant domain (He et al, Febs Letts, 1997, 450: 105).

In another aspect the present invention provides a method comprisingsteps (a), (b) and (c) as indicated, wherein the translation system iseukaryotic and protein disulphide isomerase (PDI) is employed in theincubation conditions. PDI is available from Sigma and Pierce.

Experimental examples included below, demonstrate that use of PDIincreases the amount of correctly folded specific binding memberavailable to bind its cognate complementary binding molecule, and thatthe conditions when included in a modified ribosome display selectionsuccessfully generate antigen-specific antibodies.

In another aspect the present invention provides a method comprisingsteps (a), (b) and (c) as indicated, wherein protein disulphideisomerase (PDI) is employed in the incubation conditions, along withoxidised and reduced glutathione at a ratio of 1:1 and 10:1, in aeukaryotic or prokaryotic system.

In another aspect the present invention provides a method comprisingsteps (a), (b) and (c) as indicated, wherein oxidised and reducedglutathione is added after completion of initial translation, preferablyabout or after 30 minutes after translation initiation. This theinventors have found provides an improvement over addition atintitiation of translation.

In another aspect, the present invention provides a method comprisingsteps (a), (b) and (c) as indicated and further comprising selecting forcomplexes comprising a specific binding member able to bindcomplementary specific binding member of interest, while blockingunspecific selection using heparin. Example 7 below shows that includingheparin as a blocking agent, especially in conjunction with use of yeastt-RNA, results in an improved level of recovered RNA.

In another aspect, the present invention provides a method comprisingsteps (a), (b) and (c) as indicated, wherein the mRNA further comprisesa sequence for encapsidation of the mRNA in a viral coat. On provisionof viral coat protein that recognises the sequence for encapsidation,the complex of mRNA, ribosome and displayed specific binding member isencapsidated in the viral coat protein.

The viral coat protein and OAS may be TMV (Durham (1972) J. Mol.

Biol. 67, 289-305; Goelet et al., (1982) PNAS USA 79, 5818-5822). Otherviral coat proteins such as those from the following virus families:Tobamovirus, Potexvirus, Potyvirus, Tobravirus, Cucumovirus orComovirus, Togaviridae, Flaviviridae, Picornaviridae and Caliviridaealong with their cognate packaging sequences may be used. Coat proteinsderived from DNA viruses such as cauliflower mosaic virus may also beemployed for encapsidating DNA or RNA, as can coat proteins ofbacteriophages.

The viral coat protein may be provided prior to translation orco-translationally.

Incorporation of the OAS into the ribosome display construct allows theviral coat protein to nucleate encapsidation of the RNA construct. Coatprotein is provided in a form suitable to initiate encapsidation. Thisform is preferably a “disc” preparation, as described in Durham (1972)J. Mol. Biol. 67, 289-305. It has been demonstrated in examplesdescribed below that an OAS can be inserted into the ribosome displayconstruct and that RNA derived from this construct can be encapsidatedin TMV coat protein. The encapsidated transcript can be translated invitro to generate scFv of the appropriate size, hence packaged RNAretains the ability to be translated. Encapsidation of a single speciesof foreign (i.e. non TMV) RNA has been demonstrated by Sleat et al.,(1986) Virology 166, 209-308, although encapsidation of populations ofRNA has not been reported in the literature. It has been demonstratedthat TMV is translated by a process of co-translational uncoating inwhich the plant ribosome simultaneously strips off the viral coatprotein whilst translating the viral genome (Wilson (1985) J. Gen.Virol. 66, 1201-1207). This process of co-translational uncoating hasalso been shown for “pseudovirus” particles of foreign RNA produced inplants (Plaskitt et al., (1998) Plant-Microbe Interactions 1, 10-16),and is the likely mechanism of translation of the encapsidated ribosomedisplay constructs. The presence of a ribosome on the OAS-containing RNAdoes not necessarily inhibit encapsidation of that RNA. It has beenshown in an E. coli TMV coat protein expression system thatencapsidation of the RNA can occur up to the position of the ribosome,at which point assembly is blocked (Hwang et al., (1994) PNAS USA 91,9067-71).

Example 4 demonstrates successful packaging of mRNA/ribosome complex.Further addition of viral coat protein following translation may be usedto repackage mRNA/ribosome/polypeptide complex to provide furtherstability.

In a related aspect, the present invention provides a nucleic acidconstruct (DNA or RNA) comprising the following elements RNA polymerasebinding site, Kozak consensus sequence, ribosome binding site,initiation codon, coding sequence, tether sequence and OAS. One or moreadditionally features may be included.

In a further aspect the present invention provides a library orpopulation of RNA molecules, each RNA molecule in the library orpopulation containing a viral OAS and a sequence encoding a polypeptideor peptide specific binding member such as an antibody molecule, whereinthe library or population collectively encodes a population orrepertoire of specific binding members of diverse sequence. In preferredembodiments one or more additional features for ribosome display isincluded in the RNA molecules.

A library or population of RNA molecules according to the presentinvention may be packaged within viral coat, so a still further aspectof the present invention provides a population of viral particles,collectively harbouring or containing a population of RNA moleculesencoding a population or repertoire of specific binding members ofdiverse sequence. Each viral particle in the population may contain RNAencoding a polypeptide or peptide of different sequence.

In addition to an origin of assembly sequence for viral coat protein,the mRNA molecules employed in a method comprising steps (a), (b) and(c) above may further comprise an MDV sequence for amplification by Qβreplicase. This enables replication of selected RNA populations withoutany necessity for a separate RT-PCR step.

In a related aspect, the present invention provides a nucleic acidconstruct (DNA or RNA) as disclosed and comprising an MDV sequence.

In a further aspect of the invention, mRNA molecules for incubation inthe translation system are provided by means of RT-PCR reactions inwhich at least one of the RT-PCR primers is a mutagenic primer encodinga diversity of different sequences for inclusion in a defined region ofthe mRNA coding region, e.g. a region encoding a CDR of an antibodymolecule, preferably CDR3 of an antibody VH domain.

Embodiments of this aspect of the invention are illustrated further inExamples 7 and 8, and in FIGS. 7, 8 and 9.

Further aspects of the present invention provide a library, populationor repertoire of DNA or RNA molecules with the features disclosed foraspects concerned with nucleic acid constructs, wherein the library,population or repertoire of DNA or RNA molecules comprise the disclosedfeatures for each of these aspects and collectively encode a diversepopulation of different polypeptides or peptides that may form specificbinding pair members.

Still further aspects provide an expression system, such as an in vitroexpression system, e.g. rabbit reticulocyte lysate or a bacterialsystem, comprising a nucleic acid construct or library, population orrepertoire thereof, especially under culture conditions for translationof encoded polypeptide or peptide from the encoding nucleic acid.

In preferred embodiments, the specific binding members for display onthe ribosomes are antibody molecules, usually single chain antibodymolecules, such as scFv antibody molecules, VH, Fd (consisting of the VHand CH1 domains), or dab molecules. Non-antibody specific bindingmembers for display in other embodiments of the present inventioninclude receptors, enzymes, peptides and protein ligands.

Following selection and retrieval of nucleic acid encoding the displayedspecific binding member, the nucleic acid may be used in provision ofthe encoded specific binding member or may be used in provision offurther nucleic acid (e.g. by means of an amplification reaction such asPCR). Nucleic acid encoding component parts of the specific binding pairmember may be used in provision of further specific binding molecules,for instance reformatted antibody molecules. Thus, for example, nucleicacid encoding the VH and VL domains of a selected scFv antibody moleculemay be used in construction of sequences encoding antibody molecules ofother formats such as Fab molecules or whole antibody.

Furthermore, nucleic acid may be subject to any technique available inthe art for alteration or mutation of its sequence. This may be used toprovide a derivative sequence. A sequence may be provided which encodesa derivative of the selected specific binding member or componentthereof, for example a derivative that comprises an amino acid sequencethat differs from the selected specific binding member or componentthereof by addition, deletion, insertion and/or substitution of one ormore amino acid sequences. A method providing such a derivative mayprovide a fusion protein or conjugate wherein an additional peptide orpolypeptide moiety is joined to the specific binding member or componentthereof, e.g. a toxin or label.

Encoding nucleic acid, whether reformatted or not, may be used inproduction of the encoded polypeptide or peptide using any techniqueavailable in the art for provision of polypeptides and peptides byrecombinant expression.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Pluckthun, A. Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of a specific binding member, see forrecent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4:573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate.

Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate.For further details see, for example, Molecular Cloning: a LaboratoryManual: 2nd edition, Sambrook et al., 1989, Cold Spring HarborLaboratory Press. Many known techniques and protocols for manipulationof nucleic acid, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, and analysis of proteins, are described in detail in CurrentProtocols in Molecular Biology, Second Edition, Ausubel et al. eds.,John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubelet al. are incorporated herein by reference.

Thus, nucleic acid encoding a specific binding member selected using amethod of the invention, or a component of such a specific bindingmember (e.g. VH and/or VL domain) may be provided in an expressionsystem for production of a product polypeptide. This may compriseintroducing such nucleic acid into a host cell. The introduction mayemploy any available technique. For eukaryotic cells, suitabletechniques may include calcium phosphate transfection, DEAE-Dextran,electroporation, liposome-mediated transfection and transduction usingretrovirus or other virus, e.g. vaccinia or, for insect cells,baculovirus. For bacterial cells, suitable techniques may includecalcium chloride transformation, electroporation and transfection usingbacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forproduction of the encoded product.

The present invention also provides a method which comprises using aconstruct as stated above in an expression system in order to express aspecific binding member or polypeptide as above.

Following production by expression, a product may be isolated and/orpurified and may be formulated into a composition comprising at leastone additional component. Such a composition may comprise apharmaceutically acceptable excipient, vehicle or carrier.

Further aspects and embodiments of the present invention will beapparent to those skilled in the art in the light of the presentdisclosure. It should further be noted that all documents mentionedanywhere herein are incorporated by reference.

The present invention will now be further illustrated with reference tothe following experimental examples.

List of Examples

Example 1—Monosome formation

Example 2—Improving ScFv refolding conditions for the ribosome displaysystem

Example 3—Generation of a naïve PCR assembled library using a tethercassette

Example 4—Generation of a packagable PCR assembled library

Example 5—Generation of a packagable library which incorporates an RNAreplicase cassette

Example 6—Generation of an affinity maturation library by PCR assembly

Example 7—An improved selection regime

Example 8—Use of the improved selection regime to select for affinitymatured variants of an antibody isolated against a GPI-linked cellsurface receptor.

Example 9—Comparison of structured versus unstructured tethers in aselection format

EXAMPLE 1

Monosome Formation

a) Introduction

Efficient ribosome display selection will probably occur if one ribosometranslates each mRNA molecule of the library, producing a singlefull-length protein molecule. If more than one ribosome translates asingle mRNA molecule the additional ribosomes will be prevented fromcompletely translating the mRNA due to the presence of the stalled,unreleased initial ribosome. The resultant translated protein moleculeswill be truncated and unable to assume correctly folded configurations.This may result in non-specific association of incorrectly foldedmaterial to the antigen in the selection process and may reduce itsefficiency. The inventors anticipated that a 1:1 ratio of ribosomemolecules to mRNA molecules may provide the most favourable circumstanceto ensure the maximal library size is translated with only one ribosomeper mRNA molecule.

b) Determination of the Number of Ribosomes Per mRNA by ElectronMicroscopy

Nucleic acid with a sequence encoding a scFv antibody known to bind toFITC was cloned into a ribosome display vector (FIG. 2A, 2B—theconstruct cloned into a pUC-based vector) either with or without aterminal stop codon. The scFv coding region was then PCR amplified usingprimers PEU and HA mini to generate DNA fragments encoding the antibodydownstream of the T7 promoter, ribosome binding site and Kozak consensussequence. These PCR products (with or without a terminal stop codon)were then used as starting templates in a coupled rabbit reticulocytetranscription translation system with biotinylated lysine included inthe translation mix to generate biotinylated scFv.

Translation reactions were set up by making a master mix of 100 μlPromega rabbit reticulocyte lysate, 2.5 μl 1 mM methionine, 2 μl PromegaTranscend tRNA and 15.5 μl sterile distilled water. 2 μl of PCR product(approximately 200 ng) was then added to 48 _l of the master mix and thereaction incubated for 1 hour at 30° C. Dilutions of the translationreaction were then immobilised on an electron microscope grid andstained with uyranyl acetate, followed by streptavidin-gold particles tolabel the biotinylated lysine residues.

In the case of the PCR fragment that included a stop codon at the end ofthe scFv gene no association of streptavidin gold with assembledribosomes was observed. This is as expected, since the stop codon wouldallow release of the ribosome and hence dissociation of the antibodyribosome mRNA (ARM) complex. In the case of the PCR product that did notcontain a stop codon the ARM complex remained intact, since the ribosomewas not released. In this case the presence of streptavidin-goldparticles in association with the assembled ribosomes was observed. Theassembled ribosomes were observed in isolation, rather than in groups orlines. This is compelling evidence that monosomes rather than polysomeswere generated by the translation process using the ribosomeconcentration and DNA template concentrations employed in theexperiment.

EXAMPLE 2

Improving ScFv Refolding Conditions for the Ribosome Display System

a) Introduction

Much of the initial work on polysome display was carried out usingpeptide libraries in which protein folding conditions are not critical.The expression of larger protein or polypeptide libraries, such asantibody fragments, is potentially more dependent on theoxidation/reduction environment in which the protein is translated. Theprotein is folded as it is synthesised in eukaryotic systems andvariation of the ratio of oxidised to reduced glutathione cansignificantly effect the efficiency of the folding process. Chaperonesand protein disulphide isomerase (PDI) are more likely to be effectivein prokaryotic systems because the newly synthesised protein is releasedfrom the ribosome prior to folding. In contrast, eukaryotic systemscontain endogenous PDI (Ryabova et al, 1997, Nature Biotechnology, 15:79-84) and no benefit to adding PDI to such a system could be expected.The present inventors have however shown that use of PDI, and use of PDIin combination with ox:red glutathione results in improved levels ofrecovered correctly folded specific binding members.

b) Effect of Variation in the Redox Potential of the In VitroTranslation Reaction on scFv Activity

An assessment of the proportion of correctly folded scFv antibodyfragments was made by subcloning a panel of existing scFv gene fragmentsthat had been isolated by phage display (Table 1) into the ribosomedisplay vector (FIG. 2A, 2B). These antibodies were then in vitrotranscribed and translated using a coupled rabbit reticulocytetranslation mix. ³⁵S-methionine was included in the translation mix andproduction of active antibody was assayed by binding of theradiolabelled scFv to antigen. In vitro translations were set up bymaking a master mix of 100 μl TNT lysate, 2.5 μl ³⁵S-methionine and 17.5μl sterile distilled water. 2 μl of PCR product was then added to 48 μlof master mix and the reaction incubated for 60 min at 30° C. Oncompletion of the reaction samples were treated with an equal volume (50μl) of RNase and incubate at room temperature for 15 min. The sampleswere then blocked with 20 μl of 18% Marvel in 6×PBS for 10 min. ELISAplates were coated with the appropriate antigen at 1 μg/ml at 4° C.overnight, then blocked for 1 hour at 37° C. with 200 μl of 3% Marvel inPBS. Half the blocked translation reaction was added to theantigen-coated wells and half to a well coated with a control protein,and allowed to bind for 1 hour at 37° C. After the incubation the liquidwas removed using a pipette and the wells washed with 200 μl PBScontaining 0.1% Tween, followed by three washes with 200 μl PBS. BoundscFv was eluted by adding 50 μl of 100 mM triethylamine (TES) to eachwell, and transferring this to a separate scintillation plate containing25 μl of 1M Tris (pH 7.4) to neutralise the TEA. 100 μl of scintillationfluid was then added and the sample read on Top Count. Using thestandard in vitro translation conditions of the commercially availabletranslation kit 4/12 of the antibodies expression gave a detectablesignal in the binding assay. The ratio of oxidised:reduced glutathionepresent in the translation mix was then varied and the panel of scFvantibodies tested in the binding assay under the new conditions. Ratiosof: 10:1 oxidised:reduced; 1:1 oxidised:reduced; 1:10 oxidised:reducedand reduced only were included in the in vitro translation mix and wereadded after the reaction has proceeded for 30 min. A 1:1 ratio ofoxidised: reduced glutathione involved adding 50 μl of 4 mM oxidisedglutathione and 4 mM reduced glutathione. A typical example of theeffect is shown in FIG. 3. At oxidised:reduced glutathione ratios of10:1 and 1:1 the percentage of radioactivity labelled anti-TNF antibodywhich bound to the antigen was significantly increased. This trend ofincrease in active scFv at the 10:1 and 1:1 ratios held for allantibodies tested.

c) Assessment of the Effect of Addition of the Chaperone ProteinDisulphide Isomerase on scFv Activity

The panel of scFv antibodies cloned into the ribosome display vectorwere assessed for binding to antigen in ELISA format by the capture ofradiolabelled antibody. Translation mixes were set up as describedabove. An oxidised:reduced glutathione ratio of 1:1 was included in thetranslation mix and protein disulphide isomerase (PDI) was added at arange of concentrations from 0 to 200 μg/ml. It was found that PDI addedat a concentration of 10-20 μg/ml enhanced the signal obtained in thecapture assay. Example results showing the effect of PDI on scFvactivity appear in FIG. 4.

The time of addition of PDI to the translation mix was also assessed.PDI was added at 0, 10, 20 or 30 minutes after the start of thetranslation reaction, and little change in captured scFv was observed,suggesting the time of addition is not critical.

d) Summary of Improved Scfv Folding Conditions In Vitro TranslationReactions.

In vitro translation reactions for the production of greater amounts ofcorrectly folded scFv have been determined to be the inclusion of about20 μg/ml PDI at time zero in the reaction mix. After the reaction hasproceeded for 30 mins 4 mM GSSG, and 4 mM GSH are added (1:1 oxidised:reduced glutathione) and the reaction is continued for a further 30 min.Reaction temperature is 30° C. Under these conditions the number of scFvwhich gave detectable binding in the capture assay increased from 4/12under standard translation conditions to 7/12. These data are summarisedin Table 2. The specificity of none of the scFv tested was altered bythe modified translation conditions.

EXAMPLE 3

Generation of a Naïve PCR Assembled Library Using a Tether Cassette

a) Introduction

For an effective ribosome display repertoire to be produced a tether isincluded to provide a spacer between the ribosome and the displayedpolypeptide.

Tethers used to date described in the literature have comprisedfragments of naturally occurring structured proteins such as the geneIII protein of the filamentous bacteriophage Fd (Hanes and Pluckthun,PNAS USA 1997, 94, 4937-4942) or antibody constant domains (He andTaussig, NAR, 1997, 25, 5132-5134). We have incorporated a syntheticglysine-serine tether into the ribosome display construct which willhave little integral secondary structure, as compared to the tethersalready described. This may be used to reduce the stringency of thefolding conditions required for efficient ribosome display of a givenprotein and provide more flexibility in the tether region, reducingpossible stearic hindrance effects between the ribosome and theexpressed protein. The tether fragment may vary in length from 2 aminoacids to about 400 amino acids, e.g. one glycine-serine unit to up toabout 200 glycine-serine units, and may encode other peptides orproteins which have limited secondary structure. It is also possible touse the tether cassette as a way of incorporating other types of encodedfunction into the the ribosome display repertoire. For example sequencesencoding packaging signals, or RNA replicase sequences may also beincluded.

b) Generation of PCR Assembled Library

A scFv antibody repertoire was PCR amplified from an expanded version ofthe phage display scFv library cloned into pCantab6 (Vaughan et al1996). PCR was carried out using the primers PEU and mycseq10 using 30cycles of 94° C. 1 min, 55° C. 1 min, 72° C. 2 min, and the resultantPCR product was gel purified. A tether fragment was produced by PCR ofthe RDV-stuffer vector (FIG. 2A, 2B) using primers hismyc Back and HAtag using the same PCR conditions as the scFv. Assembly and pull throughreactions were carried out using the primers T7 and HA mini (AppendixII). Assemblies were carried out using 25 cycles of 94° C. 1 min, 55° C.4 min. One tenth of the assembly reaction (5 μl) was then added to apull through reaction and PCR amplified using 30 cycles of 94° C. 1 min,55° C. 1 min, 72° C. 2 min. The pull through reaction is a PCR that usesprimers which are at the extreme ends of the two DNA fragments beingannealed in the assembly reaction. In this way, full length assembledproduct is amplified from the fragment mixture. An assembled product ofthe expected size (1.1 kb) was produced and gel purified. This productcan be used directly as starting template for a coupled in vitrotranslation/transcription reaction.

Primers used (all written 5′-3′): PEU (SEQ ID NO: 2) AA TTC TAA TAC GACTCA CTA TAG GGA GAG CAC TTC TGA TCC AGT CCG ACT GAG AAG GAA GGC CCA GCCGGC CAT GG HA TAG (SEQ ID NO: 3) TAC CCG TAT GAC GTG CCG GAT TAC GCA T7(SEQ ID NO: 4) TAA TAC GAC TCA CTA TAG GGA GAG CAC TTC TG HA mini (SEQID NO: 5) TGC CTA ATC CCC CAC Mycseq 10 (SEQ ID NO: 6) CTC TTC TGA GATGAG TTT TTG Hismyc back (SEQ ID NO: 7) GCA CAT CAT CAT CAC CAT CAC GGGGCCC) Characterisation of the PCR Assembled Library on the Basis of scFvExpression

The scFv repertoire assembled with a glycine-serine tether was used astemplate in an in vitro translation reaction using the conditionsdescribed in Example 7. The translation reaction was run out on aprotein gel and western blotted. ScFv was detected by probing the blotwith an anti-myc tag antibody, followed by and anti-species HRPconjugate. Levels of scFv production from the assembled library wereestimated to be between 50-100 μg/ml.

d) Analysis of Sequence Diversity of the Unselected PCR AssembledLibrary

A fraction of the library was digested with the restriction enzyme BstNi which has a frequently occurring four residue recognition sequence.On digestion of the library with this enzyme a ladder of bands resulted,demonstrating that the library consists of a mixed population of scFvgene segments. When the library was digested with Sfi I a single bandwas observed of the expected size. A fraction of the library was alsodigested with Sfi I and Not I and cloned into the ribosome displayvector to allow sequencing of individual scFv gene fragments present inthe library. 48 scFv fragments were sequenced and all were found todifferent. These data provide indication that the population of scFvgene segments present in the PCR assembled library is diverse.

EXAMPLE 4

Generation of a Packagable PCR Assembled Library

Incorporation of TMV OAS into constructs for ribosome display libraries.

The core positions of the OAS correspond to positions 5420-5546 of theTMV RNA sequence (Goelet et al., 1982, PNAS USA 79, 5818).

A library of scFv fragments was generated by PCR amplification, asdescribed (Example 3). Polyhistidine and myc tags were retained in thePCR fragments 3′ to the scFv coding region. An origin ofassembly-containing PCR fragment was generated by the ligation of twooligonucleotides as follows.

Oligonucleotides HA-OAS1 and HA-OAS2 were assembled together by theaddition of 2 μl (approximately 100 ng) of each oligo to 24 μl 1×TAQbuffer containing 1.5 μl of 5 mM dNTPs and 0.5 μl TAQ polymerase. Theassembly reaction conditions were 94° C. for 1 min, followed by 55° C.for 4 min in 6 cycles. A pull-through reaction was set up consisting of10 μl of the assembly reaction added to 5 μl of scFv repertoire(approximately 500 ng which had been PCR amplified with PEU and mycseq,Example 3), 5 μl 5 μM dNTPs, 5 μl 10×PCR buffer, 2.5 μl of HAmini primer(10 μM), 2.5 μl PEU (10 μM), and 0.5 μl TAQ. PCR conditions were 25cycles of 94° C. 1 min, 55° C. 1 min, 72° C. 2 min. After pull-throughreactions were complete a band of approximately 1.1 kb corresponding toassembled scFv and OAS tether was visible after gel electrophoresis.HA-OAS 1 (135 mer) (5′-3′) (SEQ ID NO: 8): TGC GTA ATC CGG CAC GTC ATACGG GTA ACT ATT TTT CCC TTT GCG GAC ATC ACT CTT TTT TCC GGT TCG AGA TCGAAA CTT TGC AAG CCT GAT CGA CAT AGG GAC ATC TTC CAT GAA CTC ATC AAC GACTTC TTC HA-CAS 2 (no stop) (144 mer) (5′-3′) (SEQ ID NO: 9): GAA CTC ATCAAC GAC TTC TTC TGT AAG TTC CAT GGG CCC TCC GTC TCT CAC GTT TGT AAT CTTCTC TCT CAA ACC ATT CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC TGC GGC CCCGTG ATG GTG ATG ATG ATG TCG GGC CGC

A version of primer OAS 2 was also produced which incorporated a stopcodon at the end of the myc tag. This oligonucleotide allows productionof OAS-containing constructs which will not have the ability to formARMs complexes because the presence of the stop codon will result inrelease of the ribosome. HA-OAS 2 stop (5′-3′) (SEQ ID NO: 10): GAA CTCATC AAC GAC TTC TTC TGT AAG TTC CAT GGG CCC TCC GTC TCT CAC GTT TGT AATCTT CTC TCT CAA ACC CTA ATT CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC TGCGGC CCC GTG ATG GTG ATG ATG ATG TCG GGC CGCc) RNA Transcription

RNA was generated by in vitro transcription of the PCR product. Atranscription reaction was assembled by the addition of approximately 4μg of PCR product (in 20 μl water) to 10 μl transcription buffer, 25 mMrNTPs, and 5 μl Promega T7 enzyme mix. The reaction was incubated for 2hours at 37° C. On completion of the reaction Dnase I was added and thereaction incubated for 15 min at 37° C. The transcription reaction wasthen phenol/choloroform extracted and divided into 4 aliquots of 12.5μl. 37.5 μl of water was added to each aliquot and the RNA then ethanolprecipitated by the addition of 5 μl of 3M sodium acetate, 1 μl glycogenand 125 μl 100% ethanol. Precipitation was carried out at B70° C. for 30min, and the RNA then pelleted by centrifugation at 13 000 rpm for 10min in a microfuge. Pellets were washed in 70% ethanol and resuspendedin 50 μl water. RNA was stored at B70° C.

d) Preparation of TMV Coat Protein

The method of preparation of TMV coat protein was based on thatdescribed by Durham, 1972 J Mol Biol 67 289-305. The method involvesdialysis of TMV in a high pH buffer (pH11) to disaggregate the coatprotein from the viral RNA. This is followed by dialysis at pH 8 andcapture of the free RNA on a DEAE-cellulose column. The protein is theneluted from the column in a small volume and dialysed in pH 5 buffer togive a disc preparation of the coat protein.

0.5 ml of 10 mg/ml U1 strain TMV was dialysed overnight at 4° C. in 0.1Methanolamine containing 0.005M HCl. Virus was dialysed for 4 hr against0.012M Tris/0.01M HCl, and the degraded virus centrifuged at 150 000 gfor 1 hr. The supernatant was loaded onto a 1 ml DEAE cellulose columnwhich had been pre-equilibrated with 0.12M Tris/0.01M HCl, and the coatprotein was eluted with 0.12M Tris/0.1 M HCl. 1 ml fractions werecollected and the bulk of the protein was collected in fraction 2. Thecoat protein was then dialysed for a minimum of 48 hr at 4° C. in sodiumacetate I=0.1, pH5.

e) Packaging Reactions.

Packaging reactions were carried out as described by Sleat et al, 1986Virology 155, 299-308. A protein:RNA ratio (w/w) of 50:1 was chosen forthe reactions. Primary packaging reactions were set up using 7 μg oftranscribed RNA and 350 μg of coat protein preparation. Total reactionvolume was 1 ml made up with 0.1 M Tris HCl pH 8, and incubation was for2 hr at room temperature. After packaging was complete the reactionswere stored at 4° C.

The success of the packaging reactions was assayed by electronmicroscopy. Samples from the original virus preparation, the coatprotein preparation and the packaging reactions were viewed in theelectron microscope after negative staining with 1% (w/v) uranylacetate. The original virus and in vitro transcribed RNA packingreaction gave clearly visible rods, the in vitro packaged RNA generatingshorter rods than the parental virus. No rods could be seen in theprotein preparation.

This demonstrates encapsidating RNA to improve stability of the RNAduring long term storage and during the selection process. EncapsidatedRNA can be directly translated in vitro in a process calledco-translational disassembly to allow generation of the ARM complex.Further viral coat protein may be added to the in vitro translationreaction after co-translational disassembly has occurred to allowrepackaging of the polypeptide/ribosome complex with the polypeptidestill displayed. This would generate a stable complex the RNA of whichwould be less prone to degradation than the unencapsidated RNA.

It is possible to express TMV coat protein in E. coli along with theOAS-containing RNA to generate in vivo packaged pseudovirus particles(Hwang et al., Proc. Natl. Acad. Sci. USA 91, 9067-9071). This may beused to provide a means to co-express TMV coat protein and mRNA encodingan OAS-containing scFv library to generate a packaged library in vivo.

EXAMPLE 5

Generation of a Packagable Library which Incorporates an RNA ReplicaseCassette

a) Design of Replication Sequence Cassette

Midivariant (MDV) RNA is a template for the RNA-directed RNA polymeraseQβ replicase (Wu et al., Proc. Natl. Acad. Sci. 89, 1992 11769-73). TheMDV RNA consists of two separate regions of RNA which hybridise togetherto form a distinct secondary structure which enables the Qβ replicase torecognise the RNA and catalyse its exponential amplification. Thepresent inventors have included the sections of MDV RNA in a ribosomedisplay construct that generates RNA that can be replicated in vitro.Such a construct may also include the TMV or other viral OAS packagingsequence to allow encapsidation of the resultant RNA molecules. Thedesign of a ribosome display construct incorporating MDV and OASsequences is shown in FIG. 6.

Primers to allow the incorporation of MDV RNA into the ribosome displayconstruct are shown below:

The MVD1 replication site includes 63 nucleotides at the 5′ end of theconstruct as follows (5′-3′): (SEQ ID NO: 11)GGGGACCCCCCCGGAAGGGGGGGACGAGGTGCGGGCACCTCGTACGGGAG TTCGACCGTGACG.

This 63 nucleotide segment is then followed by the expression unitcontaining the scFv gene segments, detection and purification tags, theTMV OAS sequence if required and a tether. The 3′ end of the constructthen includes the 3′ MDV sequence that is 156 nucleotides long asfollows (5′-3′): (SEQ ID NO: 12)CACGGGCTAGCGCTTTCGCGCTCTCCCAGGTGACGCCTCGTGAAGAGGCGCGACCTTCGTGCGTTTCGGTGACGCACGAGAACCGCCACGCTGCTTCGCAGCGTGGCTCCTTCGCGCAGCCCGCTGCGCGAGGTGACCCCCCGAAGGGGG GTTCCC.

The 3′ segment of the MDV sequence is too long to be made as acontinuous oligonucleotide, so is split into two overlapping segmentswhich can be made as single oligonulcoetides which can be annealedtogether. Three MDV oligonucleotides in total are required as follows:

MVD1 (encoding the 5′ 63 nucleotides of the MDV sequence followed by 23nucleotides of the T7 promoter shown in bold) (5′-3′). (SEQ ID NO: 13)GGGGACCCCCCCGGAAGGGGGGGACGAGGTGCGGGCACCTCGTACGGGAGTTCGACCGTGACGAATTCTAATACGACTCACTATAG

MDV2: HA detection tag (bold face) followed by the first 79 nucleotidesof the 3′ segment of the MDV RNA.

Sense (SEQ ID NO: 14) TACCCGTATGACGTGCCGGATTACGCACACGGGCTAGCGCTTTCGCGCTCTCCCAGGTGACGCCTCGTGAAGAGGCGCGACCTTCGTGCGTTTCGGTGAC GCACGA

Reverse Complement (51-3′) (SEQ ID NO: 15)TCGTGCGTCACCGAAACGCACGAAGGTCGCGCCTCTTCACGAGGCGTCACCTGGGAGAGCGCGAAAGCGCTAGCCCGTGTGCGTAATCCGGCACGTCATA CGGGTA

MVD3: Remaining 77 nucleotides of the 3′ MDV segment within anadditional 19 nucleotide overlap (bold face) with MDV2 to allowassembly.

Sense (SEQ ID NO: 16) GCGTTTCGGTGACGCACGAGAACCGCCACGCTGCTTCGCAGCGTGGCTCCTTCGCGCAGCCCGCTGCGCGAGGTGACCCCCCGAAGGGGGGTTCCC

Reverse complement (SEQ ID NO: 17)GGGAACCCCCCTTCGGGGGGTCACCTCGCGCAGCGGGCTGCGCGAAGGAGCCACGCTGCGAAGCAGCGTGGCGGTTCTCGTGCGTCACCGAAACGCb) Assembly Conditions

MVD2 and MDV3 oligonucleotides were assembled together by the additionof 2 μl of each oligo to 24 μl 1×TAQ buffer containing 1.5 μl of 5 mMdNTPs and 0.5 μl TAQ polymerase. The assembly reaction conditions were94° C. for 1 min, followed by 55° C. for 4 min in 6 cycles. A three waypull-through reaction was set up consisting of 10 μl of the assemblyreaction, 2 μl of MDV1 oligonucleotide and 5 μl of scFv OAS repertoire(approximately 500 ng which had been PCR amplified with PEU and HA back,Example 3), 5 μl 5_M dNTPs, 5 μl 10×PCR buffer, 2.5 μl of MDV3 (10_M),2.5 μl PEU (10_M), and 0.5 μl TAQ. PCR conditions were 25 cycles of 94°C. 1 min, 55° C. 1 min, 72° C. 2 min. After pull-through reactions werecomplete a band of approximately 1.3 kb corresponding to the fullyassembly product was gel purified. This DNA was then digested with Sfi Iand Not I and cloned into Sfi I/Not I cut ribosome display vector,allowing transcription of the full length mRNA which could then bereplicated with Qβ replicase, or packaged in TMV CP.

EXAMPLE 6

Generation of an Affinity Maturation Library by PCR Assembly

The PCR assembly strategy described in Example 3 is applied togeneration of libraries designed for affinity maturation of a parentalscFv. The PCR primers are designed to generate the main body of parentalscFv from the first few residues of the VL CDR3 through to the start ofthe heavy chain, as shown in FIG. 7. The remaining portion of the VLCDR3 (the area to be targeted by the mutagenesis) is then generatedusing a mutagenesis primer which overlaps with the start of the VL CDR3paired with a primer terminal to the tether (e.g. HA back) as shown inFIG. 7. The two fragments are assembled and pull through using standardconditions to provide template for a ribosome display selection. Thisprocedure may be modified to generate libraries of mutants in each ofthe CDRs as shown in FIG. 8. It may also be used to sequentially mutatedifferent CDRs at each round of selection, as shown in FIG. 9.

EXAMPLE 7

An Improved Selection Regime

a) Introduction

A polypeptide which binds a complementary sbp member of interest (e.g.antibody molecule that binds antigen of interest) can be selected from alibrary of polypeptides displayed on ribosomes using the complementarysbp member (e.g. antigen) either coated onto panning tubes or insolution. Once the binding molecules are captured RNA is eluted and putinto a reverse transcriptase BPCR (RT-PCR) to generate DNA. This DNA canthen either be cloned into a ribosome display vector (or otherexpression vector) or can be re-assembled to include a proteinexpression unit and appropriate tether and can be subjected to furtherrounds of selection. This process is reliant on keeping the RNA moleculein association with the ribosome in the initial stage of the selectionand ensuring that the eluted RNA is full length so that full lengthpolypeptide-encoding DNA can be regenerated from it. An example of aprotocol used to select anti-FITC antibodies from a naive library inaccordance with various aspects of the present invention is describedbelow.

b) Selection

A panning tube was coated with 1 ml of FITC-BSA at 100 μg/ml overnightat room temperature. The next day the tube was blocked with 2 ml 10% BSAcontaining 1 mg/ml tRNA and the tube was shaken for 1 hour at roomtemperature, and then for 1 hour at 4° C.

A master translation mix was prepared by the addition of 93.7 μl TNTlysate to 2.4 μl 1 mM methionine, 2.3 μl PDI (at 20 μg/ml) and a maximumvolume of 26.6 μl PCR assembled library (1-2 μg). This mix was splitinto two reactions of 62.5 μl and incubated at 30° C. for 30 min, afterwhich an equal volume of 4 mM 1:1 oxidised:reduced glutathione(GSSG:GSH) was added. The reaction was incubated for a further 30 min at30° C. and diluted immediately into 750 μl ice-cold heparin at 2.5 mg/mlin 1×TBS containing 0.1% Tween, 5 mM MgOAc. Coated panning tubes werewashed 3-5 times with 1 ml of the heparin block solution at 4° C. Thetranslation reaction was then added to the tube and shaken gently for 1hour at 4° C. All remaining steps were carried out at 4° C. usingice-cold tips and tubes. The tubes were washed 10 times with 2 mlheparin block and the RNA then eluted with 200 μl elution buffer (20 mMEDTA, 1×TBS, RNase inhibitor at 1 U/μl). To ensure efficient elution thetubes were vortexed several times over 10 min. 100 μl PBS was then addedto the eluted sample, along with 400 μl lysis buffer from a BoehringerHigh Pure RNA Isolation kit. RNA was purified as described in the kitand eluted in 50 μl of kit elution buffer.

c) RT-PCR

RT-PCR was carried out using the purified RNA as template using an ABgene RT-PCR kit. A mix for one RT-PCR consisted of 25 μl enzyme mix, 1μl RT-enzyme, 5 μl RNA, 1 μl Bigpam primer (10 μM), 1 μl Myc37 primer(10 μM), 17 μl water. Control reactions with no added RT enzyme were setup in parallel to demonstrate absence of DNA contamination. PCRconditions were 30 cycles of 94° C. 1 min, 64° C. 1 min, 72° C. 2 min.The resultant PCR product was either cloned as a Sfi I/Not I fragmentinto RDV or pCantab6, or was reassembled into a full-length ribosomedisplay as described below.

d) Re-assembly of RT-PCR Product

100 ng gel purified RT-PCR product and 50 ng tether PCR fragment weremade up to 50 μl with water and 1 μl glycogen, 5 μl 3M sodium acetateand 150 μl 100% ethanol were added. The DNA was precipitated at B70° C.for 30 min, then pelleted in a minifuge at 13 000 rpm for 20 min at 4°C. The pellet was washed in 70% ethanol and resuspended in 25 μl water.The DNA was the transferred to strips of 0.2 ml PCR tubes and 3 μl Taqbuffer, 1.5 μl dNTPs and 0.5 μl TAQ then added. The assembly reactionwas carried out using 25 cycles of 94° C. 1 min, 65° C. 4 min. 5 μl ofthe assembled product was added to a standard PCR mix containing theprimers PEU1 and HA Back using an annealing temperature of 58° C. PCRproducts were gel purified and could then be used as input for a secondround of selection.

E) Screening of Selection Outputs

To allow screening of outputs from the various rounds of selectionRT-PCR products were disgested with Sfi I/Not I and cloned into thephage display vector pCantab6. Individual colonies resulting from thiscloning could then be picked and screened for binding to target antigensby phage ELISA, as described in Vaughan et al., 1996.

f) Characterisation of an Anti-FITC Clone Selected from the NaïvePCR-Assembled Ribosome Display Library

Phage ELISA of a population of scFv generated by two rounds of selectionof the PCR-assembled naïve ribosome display library on FITC-BSAidentified a FITC-specific scFv. The cloned had a DP50 VH germline, andDP116 VL germline. CDR3s were as follows: VH CDR3 NMVRGVGRYYYMDV (SEQ IDNO: 18) VL CDR3 CSRDSSGYHLV (SEQ ID NO: 19)

The off rate of this clone was measured by BiaCore and found to be5×10⁻³ s⁻¹.

EXAMPLE 8

Use of the Improved Selection Regime to Selection for Affinity MaturedVariants of an Antibody Isolated Against a GPI-Linked Cell SurfaceReceptor

a) Mutagenised Libraries

A parental scFv that recognised the GPI-linked cell surface receptor ofinterest was isolated from a large phage display library using standardselection techniques. The parent clone had a K_(d) of 0.02 s⁻¹, asmeasured by BiaCore analysis of FPLC purified monomeric scFv.

The VH CDR3 of the parent had the following sequence: VHNGWYALEY (SEQ IDNO: 20).

The VL CDR3 of the parent had the following sequence: NSWDSSGNHVV. (SEQID NO: 21)

Libraries in which the central five residues of either the VH or VL CDR3were mutated were generated by oligonucleotide mutagenesis and clonedinto the ribosome display vector.

Libraries were designed as follows: Library H4 (VH CDR3) VHNXXXXXEY (SEQID NO: 22) Library L4 (VL CDR3) NSWXXXXXHVV (SEQ ID NO: 23)b) Selections

RNA was transcribed from plasmid prepared from each of the librariesusing standard protocols. A typical transcription reaction was: 4 μl 5×transcription buffer (Promega); 20 units Rnasin; 4 μl of each ATP, GTP,UTP, CTP (2.5 mM); 1 μl T7 RNA polymerase; 100 ng plasmid DNA, made upto 20 μl with nuclease-free water. RNA from each library was used asinput for the first round of selection, and subsequent selections werecarried out using linear DNA as input as described in Example 7. Thefirst two rounds were carried out using the target antigen immobilisedonto plastic exactly as described in Example 7. This was performed forboth the H4 and L4 libraries. Two further rounds of selection werecarried out on the L4 library using biotinylated antigen atconcentrations of 100 nM for round 3 and 10 nM for round 4. Selectionsusing biotinylated antigen were carried out as described in Example 7(b) except that instead of adding the ARM complexes to a panning tubebiotinylated antigen was added directly to the translation mix after ithad been diluted in ice-cold heparin buffer. The mixture was thenincubated for 1 hour at 4□C, after which time biotinylated antigen alongwith associated ARM complexes was captured on streptavidin-coatedmagnetic beads (Dynal) which had been pre-blocked with heparin blocksolution (Example 7). Beads were washed, as described for the panningtubes, except they were not vortexed and after each wash the beads werepelleted on a magnet to allow removal of the supernatant. RNA was elutedfrom the beads with 200 μl elution buffer (20 mM EDTA, 1×TBS, Rnaseinhibitor at 1 U/μl), and RT-PCR, re-assembly and analysis carried outas described (Example 7).

c) Results

i) H4 Library

The output of the selections was screened initially by ELISA todetermine the percentage of clones selected that recognised the targetantigen. For the H4 library after two rounds of panning the percentageof clones that were positive for binding to the antigen was 55%. 135 ofthese positive clones were picked and sequenced and were found toconsist of 133 different sequences.

These clones were all prepared as peripreps and ranked by off rate usingBiaCore analysis. The five clones with the longest off rate asdetermined by this preliminary screen were then prepared asFPLC-purified monomeric scFv and accurate off rates determined. Resultsare shown in Table 3.

Two of the clones (B2B4 and B2H1) had improved off rates compared to theparental clone, demonstrating that the ribosome display selection regimedescribed is useful for generation of affinity-improved variants bytargeted mutagenesis.

ii) L4 Library

After four rounds of selection (2 panning and 2 using biotinylatedantigen) 15% of the selected clones were positive for binding to thetarget antigen. 96 positive clones were taken for BiaCore anlaysis fromthe fourth round, and of these the five with the longest off rates weretaken for further analysis. Results are shown in Table 4.

All five of the light chain CDR3 variants had improved off ratescompared to the parental clone, again demonstrating the successfulapplication of the ribosome display selection regime to generateimproved variants by targeted mutagenesis.

EXAMPLE 9

Comparison of Structured Versus Unstructured Tethers in a SelectionFormat

a) Introduction

The degree of secondary structure associated with the tether of theribosome display construct may influence the quantity and quality ofantibodies generated by a ribosome display selection process. To assessthis a comparison between a structured (bacteriophage gene III) tetherand an unstructured (glycine-serine repeat) tether was performed usingthe H4 library described in Example as a model system.

B) Preparation of Input Template

PCR amplified H4 DNA template was used as input. The H4 scFv repertoirewas amplified from the cloned H4 library (Example 8) using the primersmycseq10 and PEU. A glycine-serine tether was amplified from theribosome display vector (FIG. 2) by PCR using the primers hismycback andHA tag. A gene III tether was generated by PCR using primers describedin Hanes et al., 1999, FEBS letters 450, 105-110 with the addition ofHA, his and myc tags, and the gene III-containing vector pCantab6 astemplate. Assembly and pull-through reactions were carried out asdescribed in Example 3.

c) Selections

Two rounds of selection using immobilised antigen (GPI-linked cellsurface receptor) were carried out as described previously (Example 7).Output was screened initially by ELISA to assess the number of positiveclones generated by the selection process and a subset of clones weretaken on for BiaCore analysis to determine the off rates.

d) Results

After two rounds of selection the percentage of clones that werepositive for antigen binding by ELISA was 25% of the selections carriedout using the gene III tether and 34% for the selections carried outusing the GS tether. The off rates of 22 positive clones from eachselection were measured as peripreps on the BiaCore. 36% of the positiveclones from the gene III tether selection had improved off ratescompared to the parental clone, whereas 40% of those from the GSselection were improved.

These results provide indication of the value of use of a glycine-serinetether in a ribosome display selection system in generating clones thatbind antigen. A higher percentage of the clones selected using the GStether (c.f. the gene III tether) were improved in terms of off ratecompared to the parental clone. All the positive clones selected usingeither tether strategy have been sequenced and were found to bedifferent suggesting the type of tether used does not affect thediversity of clones selected. TABLE 1 Panel of scFv cloned into RDV1Levels of expression were determined by SDS-PAGE analysis of ³⁵S- Metlabelled protein ScFv K_(D) (nM) Antigen Expression level 1 8 TGFβ-1 ++2 2 TGFβ-2 ++ 3 0.3 TNFα ++ 4 3.7 Estradiol ++ 5 0.4 IL-12 +++ 6 2.0IL-12 +++ 7 200 IL-12 +++ 8 2000 IL-12 +++ 9 3 Fluorescein +++ 10 ?Fluorescein ++ 11 ? Fluorescein + 12 ? Fluorescein ++

TABLE 2 Activity of the panel of scFv before and after adjustment offolding conditions Activity Activity -no refolding modified ScFv K_(D)(nM) Antigen modifications folding 1 8 TGFβ-1 − + 2 2 TGFβ-2 − − 3 0.3TNFα − + 4 3.7 Estradiol − − 5 0.4 IL-12 + + 6 2.0 IL-12 − + 7 200 IL-12− +/− 8 2000 IL-12 − − 9 3 Fluorescein + + 10 ? Fluorescein + + 11 ?Fluorescein + + 12 ? Fluorescein − −

TABLE 3 Fold Mutagenesised sequence improvement Clone (VH CDR3)K_(d)(s-¹) over parent Parent GWYAL (SEQ ID NO: 24) 0.0203 — B1B3 VNLLV(SEQ ID NO: 25) 0.0233 0.87 B1F12 RSMDG (SEQ ID NO: 26) 0.0283 0.71 B2B4HAARR (SEQ ID NO: 27) 0.0113 1.79 B2H1 RVRLL (SEQ ID NO: 28) 5.9e-3 3.44B2B3 FLSSI (SEQ ID NO: 29) 0.0228 0.89

TABLE 4 Mutagenesised sequence Fold Clone (VH CDR3) K_(d)(s-¹)improvement Parent DSSGN (SEQ ID NO: 30) 0.0203 — C5 SATHE (SEQ ID NO:31) 0.0166 1.2 C10 APHGS (SEQ ID NO: 32) 0.0144 1.4 A12 TVNHD (SEQ IDNO: 33) 0.0104 2.0 D1 HWQTD (SEQ ID NO: 34) 7.4e-3 2.7 H7 NTSVT (SEQ IDNO: 35) 2.5e-3 8.12

1. A method of obtaining a specific binding pair (sbp) member that bindsa complementary sbp member of interest, the method comprising: (a)providing mRNA molecules, each mRNA molecule comprising a nucleotidesequence encoding a specific binding pair member and lacking an in-framestop codon; (b) incubating the mRNA molecules under conditions forribosome translation of the mRNA molecules to produce encoded specificbinding pair member, whereby complexes each comprising ribosome, mRNAand encoded specific binding pair member displayed on the ribosome areformed; (c) bringing the complexes into contact with the complementarysbp member of interest, and selecting one or more complexes displayingspecific binding pair member able to bind the complementary sbp memberof interest under the conditions of the selection; wherein the mRNAmolecules are incubated with prokaryotic ribosomes in a prokaryoticribosome display system or are incubated with eukaryotic ribosomes in aeukaryotic ribosome display system; and wherein mRNA molecules forincubation in the translation system are provided by means of RT-PCRreactions in which at least one RT-PCR primer is a mutagenic primerencoding a diversity of different sequences for inclusion in a definedregion of the nucleotide sequence encoding a specific binding pairmember.
 2. The method according to claim 1 wherein the mRNA moleculesincorporate a Midvariant (MDV) RNA template enabling replication by Qβreplicase.
 3. The method according to claim 1 wherein a gly-ser tetheris fused C-terminally to specific binding pair member.
 4. The methodaccording to claim 3 wherein the gly-ser tether comprises 24glycine-serine units.
 5. The method according to claim 1 whereinoxidised and reduced glutathione is added at a ratio of between 1:1 and10:1 after 30 minutes of ribosome translation.
 6. The method accordingto claim 1 wherein protein disulphide isomerase (PDI) is employed in theincubation conditions, along with oxidised and reduced glutathione at aratio of 1:1 and 10:1.
 7. The method according to claim 1 wherein thetranslation system is eukaryotic and protein disulphide isomerase (PDI)is employed in the incubation conditions.
 8. The method according toclaim 1 comprising selecting for complexes comprising a specific bindingmember able to bind complementary specific binding member of interest,while blocking unspecific selection using heparin.
 9. The methodaccording to claim 1 further comprising retrieving mRNA from a complexselected in step (c).
 10. The method according to claim 9 comprisingemploying a mutagenic primer in RT-PCR generation of a DNA copy of theribosome display library prior to a further round of selection.
 11. Themethod according to claim 9 wherein mRNA retrieved from a selectedcomplex displaying a specific binding pair member (a “selected specificbinding pair member”) is amplified and copied into DNA encoding theselected specific binding pair member.
 12. The method according to claim11 wherein the DNA is provided in an expression system for production ofa product, which product is the selected specific binding pair member ora polypeptide chain of the selected specific binding pair member. 13.The method according to claim 12 further comprising isolating orpurifying the product.
 14. The method according to claim 13 furthercomprising formulating the product into a composition comprising atleast one additional component.
 15. The method according to claim 14wherein DNA encoding the selected specific binding pair member or apolypeptide chain of the selected specific binding pair member isprovided within a nucleotide sequence to provide a nucleotide sequenceencoding a fusion protein comprising the selected specific binding pairmember, or a polypeptide chain of the selected specific binding pairmember, fused to additional amino acids.
 16. The method according toclaim 15 wherein the selected specific binding pair member comprises anantibody VH and/or antibody VL domain and the additional amino acidscomprise an antibody constant domain.
 17. The method according to claim15 wherein DNA comprising said nucleotide sequence encoding said fusionprotein is provided in an expression system for production of a product,which product is the fusion protein.
 18. The method according to claim17 further comprising isolating or purifying the product.
 19. The methodaccording to claim 18 further comprising formulating the product into acomposition comprising at least one additional component.
 20. The methodaccording to claim 11 wherein DNA encoding the selected specific bindingpair member or a polypeptide chain of the selected specific binding pairmember is mutated to encode a polypeptide that comprises an amino acidsequence that differs from the selected specific binding pair member orpolypeptide chain of the selected specific binding pair member.
 21. Themethod according to claim 20 wherein mutated DNA encoding saidpolypeptide is provided in an expression system for production of aproduct, which product is said polypeptide.
 22. The method according toclaim 21 further comprising isolating or purifying the product.
 23. Themethod according to claim 22 further comprising formulating the productinto a composition comprising at least one additional component.